Increased claudin-4 expression is associated with poor prognosis and high tumour grade in breast cancer Fiona Lanigan 1 , Eadaoin McKiernan 2,3 , Donal J. Brennan 1 , Shauna Hegarty 4 , Robert C. Millikan 5 , Jean McBryan 1 , Karin Jirstrom 6 , Goran Landberg 6 , Finian Martin 1 , Michael J. Duffy 2,3 and William M. Gallagher 1 * 1 UCD School of Biomolecular and Biomedical Science, UCD Conway Institute, University College Dublin, Dublin, Ireland 2 Department of Pathology and Laboratory Medicine, St. Vincent’s University Hospital, Dublin, Ireland 3 UCD School of Medicine and Medical Science, University College Dublin, Dublin, Ireland 4 Institute of Pathology, Queens University, Belfast, United Kingdom 5 Department of Epidemiology, University of North Carolina, Chapel Hill, NC 6 Division of Pathology, Department of Laboratory Medicine, Lund University, Malm€ o University Hospital, Malm€ o, Sweden The role of intercellular tight junctions in breast epithelial cells is traditionally thought to be in maintaining polarity and barrier function. However, claudin-4, a tight junction protein, is overex- pressed in breast tumour cells compared to normal epithelial cells, which generally corresponds to a loss in polarity. The aim of this study was to investigate the distribution and potential clinical value of claudin-4 in breast cancer, and to evaluate its usefulness as a prognostic and predictive biomarker. Expression of claudin-4 was initially examined by Western blot analysis in a cohort of 88 breast tumours, and was found to correlate positively with tumour grade and negatively with ER. Claudin-4 expression was then evaluated by immunohistochemistry in a larger cohort of 299 tumours represented on a tissue microarray. Claudin-4 expression correlated positively with tumour grade and Her2, and negatively with ER. High claudin-4 expression was also associated with worse breast cancer-specific survival (p 5 0.003), recurrence-free sur- vival (p 5 0.025) and overall survival (p 5 0.034). Multivariate analysis revealed that claudin-4 independently predicted survival in the entire cohort (HR 1.95; 95%CI 1.01–3.79; p 5 0.047) and in the ER positive subgroup treated with adjuvant tamoxifen (HR 4.34; 95%CI 1.14–16.53; p 5 0.032). This relationship between increased claudin-4 expression and adverse outcome was validated at the mRNA level in a DNA microarray dataset of 295 breast tumours. We conclude that high levels of claudin-4 protein are associated with adverse outcome in breast cancer patients, includ- ing the subgroup of patients treated with adjuvant tamoxifen. ' 2008 Wiley-Liss, Inc. Key words: breast cancer; tissue microarrays; prognostic bio- markers; claudin-4; tight junctions During progression, breast cancer tissue becomes increasingly disorganised, with associated loss of defined ductal structure and reduced ability of mammary epithelial cells to polarise. 1 These alterations are often accompanied by modified cell–cell and cell- matrix adhesions. One of the first adhesion proteins found to play a role in cancer progression was E-cadherin, the main trans- membrane protein of the adherens cell–cell junctions, which is frequently lost during cancer progression. 2 Tight junctions, the other main cell–cell adhesion complexes, are apically located and act to maintain cell polarity and control paracellular perme- ability, as well as creating a barrier between the apical and baso- lateral compartments of the plasma membrane. 3 Integral to these junctions are members of the claudin family of transmembrane proteins. The claudin family comprises 24 related members, whose expression is often tissue specific. 4 These proteins have 4 trans- membrane domains, and are considered to be the backbone of tight junctions. The extracellular loops of each claudin molecule bind to a claudin molecule on an adjoining cell at the so-called membrane ‘kissing points’. These structures, together with other integral proteins, such as occludin and junctional adhesion mole- cule (JAM), and a number of peripheral proteins, form the tight junction macromolecular complex. 5 Tight junctions can vary in composition, permeability and ion specificity depending on the tis- sue and the state of differentiation. 6 Although claudin proteins are primarily known for their cell ad- hesion function, they also interact with PDZ domain-containing molecules via a conserved YV domain at their C-terminus. 3 Thus, claudin-4 has been shown to bind to the zonula occludens (ZO) proteins, cytoplasmic tight junction components which interact with numerous signalling pathways. Claudin-4 can also bind indi- rectly to actin filaments via ZO-1 and -2, which may allow the ac- tivity of tight junctions to affect cell polarity or motility. 7–9 A large number of PDZ domain-containing proteins are now known to interact with claudins, and these are thought to be central to the intercellular signalling of tight junctions. 5 Other molecules known to interact with claudin-4 include EphA2 10 and Protein Kinase Ce, 11 both of which phosphorylate the cytoplasmic tail of claudin- 4 and regulate its incorporation into tight junctions. Several preliminary studies have been carried out on the expres- sion of different claudins in various cancer types. Although loss of claudins has been reported in some cases, such as claudin-7 in breast cancer, 12 several members of the claudin family have been found to be overexpressed in a wide variety of cancers. This includes claudin-4 in gastric cancer 13,14 and pancreatic cancer 15 ; and claudins-3 and -4 in prostate cancer, 16,17 ovarian cancer, 18 en- dometrial cancer 19,20 and breast cancer. 21,22 In breast cancer, pre- vious studies on claudin-4 have been contradictory: one study found that claudin-4 expression was lost in Grade 1 invasive breast tumours, with increased expression detected in Grade 2 and 3 tumours 22 ; others have found that claudin-4 is overexpressed in primary breast carcinomas compared to normal mammary epithe- lium. 21 Both of these studies were carried out on small patient cohorts (n 5 56 and n 5 21, respectively). The only study carried Additional Supporting Information may be found in the online version of this article. Abbreviations: BCSS, breast cancer specific survival; DCIS, ductal car- cimona in situ; EGF, epidermal growth factor; EphA2, ephrin receptor A2; ER, oestrogen receptor; Her2, human epidermal growth factor receptor 2; IHC, immunohistochemistry; JAM, junctional adhesion molecule; MMP-2, matrix metalloproteinase-2; OS, overall survival; PKC, protein kinase C; PR, progesterone receptor; RFS, recurrence free survival; TMA, tissue microarray; VEGF, vascular endothelial growth factor; VEGFR2, vascular endothelial growth factor receptor 2; ZO, zonula occludens. Grant sponsors: Enterprise Ireland, the Irish Research Council for Sci- ence Engineering and Technology under the EMBARK initiative, Cancer Research Ireland, Science Foundation Ireland, the British Association for Cancer Research and the Health Research Board of Ireland, Swedish Can- cer Society, Swegene/Wallenberg Consortium North, Gunnar, Arvid and Elisabeth Nilsson Cancer Foundation, Per-Eric and Ulla Schyberg Founda- tion, Lund University Research Funds and Malm€ o University Hospital Research and Cancer Funds. Programme for Third Level Institutions (PRTLI). *Correspondence to: UCD School of Biomolecular and Biomedical Science, UCD Conway Institute, University College Dublin, Belfield, Dub- lin 4, Ireland. Fax: 1353-1-2837211. E-mail: william.gallagher@ucd.ie Received 30 August 2008; Accepted after revision 11 November 2008 DOI 10.1002/ijc.24159 Published online 19 November 2008 in Wiley InterScience (www. interscience.wiley.com). Int. J. Cancer: 124, 2088–2097 (2009) ' 2008 Wiley-Liss, Inc. Publication of the International Union Against Cancer